Non-invasive detection and quantification of rare circulating cells in live animals is a challenging and important problem in many areas of biomedical research. Most methods rely on the extraction of small blood samples which are analyzed at a later time using techniques such as conventional flow cytometry, hemocytometry and micro-fluidic analysis. Recently, 'in vivo flow cytomety'has emerged as a technique for counting fluorescently-labeled cells in live animals without the need for extracting blood samples, but the volume of interrogation is small (~1-55L per minute) so that detection of very rare cells is difficult. Therefore, new techniques are needed for the in vivo study of very-rare circulating cell types. In this project, we propose to develop a highly-novel instrument based on the principle of fluorescence tomographic detection of fluorescently-labeled cells. The instrument utilizes a miniaturized high-speed tomographic ring design that will fit around a small limb (such as the forelimb, hindleg or tail) of an animal and allow extremely sensitive detection with blood flow rates up to 0.2 to 0.5 mL per minute, so that the entire blood volume of a mouse will be sampled in less than 10 minutes. To achieve this, the system will use: i) modulated high- intensity LEDs for fluorescence excitation, ii) high-sensitivity photon counting detection of emitted fluorescent light, iii) efficient spectral rejection of tissue and cellular autofluorescence, and iv) rapid, real-time image reconstruction at 100Hz. We will demonstrate that the instrument is capable of resolving single circulating fluorescently-labeled cells, first in a limb- mimicking flow phantom model with simulated autofluorescence, and second in BALB/c mice in vivo using two sets of fluorescently-labeled cell lines. We anticipate that the system will have applications in many areas of biomedical research including in vivo detection of metastatic tumor cells and circulating hematopoietic stem cells. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a highly novel instrument for the non-invasive detection and quantification of very rare circulating cells in live animals in vivo. The instrument will use a high-speed miniaturized tomographic ring to detect single fluorescently-labeled cells in circulation. The ring will be fitted around a limb (such as the forelimb, hindleg or tail) and will allow sampling of the entire blood volume of a mouse in less than 10 minutes, offering unprecedented detection sensitivity. We anticipate that the system will have applications in many areas of biomedical research including in vivo detection of metastatic tumor cells and circulating hematopoietic stem cells.